Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.
Procedure:
1st Day
1. Homogenize 50-500 mg of frozen post-mortem gray matter tissue with Douncing Buffer.
! CAUTION ! – Human tissue must be handled with care under strict safety conditions. It should be handled at BSL-2 or higher safety standards.
2. Micrococcal Nuclease (MN) Digestion
3. Hypotonisation
2nd Day
! CAUTION ! – Begin 2nd day by washing the Protein G Agarose that will be used to isolate nucleosomal DNA. Since agarose beads are very sensitive, it is necessary to cut off the heads of the tips whenever pipetting any solution containing agarose beads.
1. Probing Protein G Agarose Beads to DNA
2. Washing the Beads
! CAUTION ! – Washing buffers must be kept at 4°C until use, and are to be used only if are less than a month old.
* Each washing solution
— Low salt washing buffer
— High salt washing buffer
— Lithium chloride solution – only rotate at RT for 1 minute!
— TE buffer (10 mM Tris, 1 mM EDTA pH=8)
3. Elution
* CRITICAL STEP – Make fresh Elution Buffer for every experiment on the day it is to be used.
4. Digest Protein
5. Phenol/Chloroform extraction
! CAUTION ! – Experiment requiring phenol/chloroform should be performed under the hood. Use nitrile gloves when handling phenol/chloroform.
3rd Day
The protocol outlined here is particularly useful for investigators interested in histone and/or DNA methylation signatures of human brain, because these chromatin markings may be less prone to postmortem artifacts as compared to other types of modifications, including (histone) acetylation and phosphorylation1, 2. The postmortem brain is amenable to the study of mono-nucleosomal preparations; the DNA remains largely attached to the core histones, at least in specimens with representative autolysis intervals (the time between death and freezing/storing of the tissue) typically in the range of several hours up to 1.5 days1. However, mono-nucleosomal preparations could be sensitive to changes in nucleosomal positioning and densities, particularly around sequences surrounding transcription start sites of genes3. Therefore, control experiments with modification-independent anti-histone antibodies should be included. Alternatively, it may be possible to isolate poly-nucleosomal fractions from human brain extracts, using shorter incubation times for the micrococcal nuclease digest in conjunction with additional purification steps (incl. ultracentrifugation). Finally, a recent study on genome-wide transcription factor binding in postmortem brain simply sheared chromatin via sonication4. Notably, preparation of chromatin by fixation prior to sonication or enzyme-based digestion may not be ideal from the viewpoint of postmortem studies, because breakdown and/or artificial reconfiguration of higher order chromatin structures after death are potential confounds difficult to control for. Hence, ChIP assays on postmortem brain are likely to be useful for a limited number of molecules, including nucleosomal core histones and other proteins tightly attached to the genomic DNA. Even with this caveat taken into account, the approaches outlined in this presentation are likely to provide novel insights into chromatin-associated mechanisms governing neuronal and glial functions in the normal and diseased human brain.
We have used this technique successfully primarily for measuring of histone methylation and histone occupancies at specific promoters using gene by gene qPCR1, 5, 6. As stated previously the human postmortem brain seems to be amenable to the study of mono-nucleosomal preparations because the DNA remains largely attached to the core histones. Typically, when starting with 75 mg of human child or adult cerebral cortex (grey matter) one can expect using the protocol presented above- a yield of 20-30 ng/µl in a total volume of 50 µl for the Input and 10-15 ng/µl in a total volume of 50 µl for ChIP, at least when modification specific anti-histone antibodies are used. The so called ChIP to Input ratio is the unit of measure. Specificity of the reaction is monitored by melting curve analysis, gel electrophoresis, and sequencing. In addition, negative controls (i) lacking the specific antibodies or (ii) containing (non-specific) immunoglobulin should be processed by qPCR in parallel to the ChIP and Input samples and should not result in specific product. Be aware that when salmon sperm is used as a blocking agent, control samples may result in a smear when run on a gel.
This work was supported by a grant from the National Institute of Mental Health (5R01MH071476).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Tris-HCl | EMD | 9310 | ||
Magnesium Chloride Hexahydrate | OmniPur | 5980 | ||
Calcium Chloride | Fisher Scientific | C614-3 | ||
EDTA, 0.5M Solution, pH8.0 | OmniPur | 4055 | ||
Sodium Chloride | Mallinckrodt Chemicals | 7581-06 | ||
SDS Solution 10% (w/v) | Bio-Rad | 161-0416 | ||
Triton X-100 | Fluka | 93426 | ||
Igepal CA-630 | Sigma | I-3021 | ||
Sodium Deoxycholate | Sigma | D6750-25G | ||
Lithium Chloride | Sigma | L9650-100G | ||
Sodium Bicarbonate | Sigma | S7277-250G | ||
Sodium Acetate (anhydrous) | Sigma | S-2889 | ||
Nuclease micrococcal from Staphylococcus | Sigma | N3755-200UN | ||
Benzamidine | Fluka | 12072 | ||
Phenylmethanesulfonylfluoride | Sigma | P7626-1G | ||
3M DTT | Fluka | 43815 | ||
Protein G Agarose, Fast Flow | Upstate | 16-266 | ||
Sonicated Salmon Sperm DNA Kit | Stratagene | 201190 | ||
Proteinase K from Engyodontium album | Sigma | P2308 | ||
Phenol:Chloroform 1:1 | OmniPur | 6810 | ||
Glycogen, From Mussels | Sigma | G1767-1VL | ||
Ethyl Alcohol (200 Proof) | Pharmco-AAPER | 111000200 |
Solutions: